Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the mostcommon inherited disorders in humans and is causedby mutations in the NF1gene. To date, the majority ofthe reported NF1mutations are predicted to result inprotein truncation, but veryfew studieshavecorrelatedthe causative NF1mutation with its effect at the mRNAlevel. We have applied a whole NF1cDNA screeningmethodology to the study of 80 unrelated NF1 patientsand have identified 44 different mutations, 32 beingnovel, in 52 of these patients. Mutations were detectedin 87% of the familial cases, but in 51% of the sporadicones. At least 15 of the 80 NF1 patients (19%) hadrecurrent mutations. The study shows that in 50% of thepatients in whom the mutations were identified, theseresulted in splicing alterations. Most of the splicingmutations did not involve the conserved AG/GTdinucleotides of the splice sites. One frameshift, twononsense and two missense mutations were alsoresponsible for alterations in mRNA splicing. Thelocation and type of mutation within the NF1gene, andits putative effect at the protein level, do not indicateany relationship to any specific clinical feature of NF1.The high proportion of aberrant spliced transcriptsdetected in NF1 patients stresses the importance ofstudying mutations at both the genomic and RNA level.It is possible that part of the clinical variability in NF1could be due to mutations affecting mRNA splicing,which is the most common molecular defect in NF1.INTRODUCTIONNeurofibromatosis type 1 (NF1) (MIM 162200) is one of themost common inherited disorders in humans with a prevalenceof 1 in 3000 individuals. NF1 is an autosomal dominantdisorder fully penetrant at the age of 5 years, but with a vari-able clinical expression, even among members of the samefamily. The main clinical features of NF1 are cafe au lait spots(CLSs), cutaneous neurofibromas and Lisch nodules (1,2). TheNF1 gene was mapped to chromosome 17q11.2 and was posi-tionally cloned in 1990 (3–5). It spans 350 kb of genomicDNA, contains 60 exons (6,7) and is transcribed ubiquitouslyto an mRNA of 11–13 kb that encodes for a protein, neuro-fibromin, of 2818 amino acids (8). The only region of neuro-fibromin with a well-defined function and structure is a centraldomain with a high similarity to ras-specific GTPase-activating proteins (GAPs), called the GAP-related domain(GRD), which downregulates ras activity (9–11).The NF1 gene has one of the highest mutation ratesdescribed for any human disorder ( 1 10

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Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), also called von Recklinghausen disease or peripheral neurofibromatosis, is a common autosomal dominant disorder characterised by multiple neurofibromas, cafe au lait spots, and Lisch nodules of the iris, with a variable clinical expression. The gene responsible for this condition, NF1, has been isolated by positional cloning. It spans over 350 kb of genomic DNA in chromosomal region 17q11.2 and encodes an mRNA of 11-13 kb containing at least 59 exons. NF1 is widely expressed in a variety of human and rat tissues. Four alternatively spliced NF1 transcripts have been identified. Three of these transcript isoforms (each with an extra exon: 9br, 23a, and 48a, respectively) show differential expression to some extent in various tissues, while the fourth isoform (2.9 kb in length) remains to be examined. The protein encoded by NF1, neurofibromin, has a domain homologous to the GTPase activating protein (GAP) family, and downregulates ras activity. The identification of somatic mutations in NF1 from tumour tissues strongly supports the speculation that NF1 is a member of the tumour suppressor gene family. Although the search for mutations in the gene has proved difficult, germline mutation analysis has shown that around 82% of all the fully characterised NF1 specific mutations so far predict severe truncation of neurofibromin. Further extensive studies are required to elucidate the gene function and the mutation spectrum. This should then facilitate the molecular diagnosis and the development of new therapy for the disease.

https://jmg.bmj.com/content/jmedgenet/33/1/2.full.pdf

Molecular genetics of neurofibromatosis type 1 (NF1).

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

https://www.cell.com/ajhg/pdf/S0002-9297(07)64009-9.pdf

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Neurofibromatosis 1 (NF1) is an autosomal dominant neurocutaneous disorder with an incidence of approximately 1 in 4000. Cognitive deficits and academic learning difficulties are the most common neurological 'complication' of NF1 in childhood and can be responsible for significant lifetime morbidity. The NF1 gene is usually classified as a tumor suppressor gene, but it is not yet known how NF1 gene mutations cause many of the non-tumor manifestations of the disorder. The NF1 protein, neurofibromin is expressed early during embryonic development with high levels of expression in the brain, suggesting that it plays an important role in regulating the orderly differentiation of central nervous system neurons. The mouse model for NF1 demonstrates behavioral abnormalities which bear striking similarity to the cognitive phenotype observed in humans with NF1. This review summarises our current understanding of the function of the NF1 gene, the nature of cognitive deficits in this disorder and correlations between neuroradiological, pathological and neuropsychological findings and animal studies which provide an insight into the pathogenesis.

Neurofibromatosis type 1.

The neurofibromatosis 1 gene seems to play essential roles at several different stages of life. During embryogenesis, it is involved in cardiac development while in the adult, neurofibromin (the corresponding protein) is mainly expressed in the nervous system, and therein, essentially in neurons, non-myelinating Schwann cells and oligodendrocytes. In addition, the NF1 gene is considered a tumor suppressor gene, since mutations have been associated with the occurrence of benign and malignant tumors in neuralcrest-derived tissues. Using reverse transcription-polymerase chain reaction (RT-PCR) analyses with primers located in exons 7 and 13, we have identified evidence of alternative splicing in this region of the NF1 gene. Cloning and sequencing of cDNA allowed the characterization of an isoform bearing an extra 30 bp sequence between exons 9 and 10a, leading to the insertion of 10 amino acids between residues 420 and 421 of neurofibromin. The insertion is conserved in the mouse. Examination of the pattern of expression of this isoform demonstrated a high level of expression in the central nervous system and an absence of expression in all the other normal tissues tested including peripheral nervous tissues derived from the neural crest. Analysis of brain tumors indicated a reduced expression of the alternative exon in medulloblastomas and oligodendrogliomas. The results presented here are consistent with tissue-specific expression of this alternative exon which we propose to call exon 9br.

D Fauvet

Papers

Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5′ part of the gene